Steroids 2,4-dinitrophenylhydrazones

The analysis of keto steroids as their 2,4-dinitrophenylhydrazone (DNPH) derivatives by TLC [30] and HPLC [31,32] is a sensitive and reliable method for the determination of these compounds in urine and in other biological fluids. The derivatives are easily separated by TLC or HPLC and can be detected in quantities as low as 1 ng. Several variations of the reaction procedure may be used. Two of these are described below. 

Dinitrophenylhydrazones of carbonyl compounds may be formed as described for keto steroids (Section 4.1.1.4.1) by precipitation or extraction from aqueous perchlorate as described by Neuberg et al. [65], by the method of Houben-Weyl [66] or by the method of Shriner et al. [67] using sulfuric acid—water-ethanol as the reaction medium. 

It also has been used as a spray reagent and for quantitation in paper and thin layer chromatography Section 6.5). It should be noted that A - 4-3-keto steroids react much less readily with isonicotinic acid hydrazide than do A4-3-keto steroidsThe 2,4-dinitrophenylhydrazone has also been described4. 

Special techniques have been used for the measurement of individual steroids. For blood progesterone Harbert et al. (H4) obtained an initial separation on an alumina column, but final purification and assay was achieved by further alumina chromatography after preparing the bis-dinitrophenylhydrazone. The method of Short (S16) involving paper chromatography and assay by ultraviolet absorption, with Allen corrections for impurity, has been used by Greig et al. (G5) and Aitken et al. (A3). Cortisol has been measured in urine and blood by fluorimetry and isotope dilution (U3, U4), and in blood by double isotope dilution derivative techniques together with cortisone (H9) and also with cortisone,... 

Sevanian et al. (1994) applied GLC and LC/TS/MS for the analysis of plasma cholesterol-7-hydroperoxides and 7-ketocholesterol. Analysis of human and rabbit plasma identified the commonly occurring oxidation products, yet dramatic increases in 7-ketocholesterol and cholesterol-5p, 6P-epoxide were observed. The study failed to reveal the presence of choles-terol-7-hydroperoxides, which were either too unstable for isolation, metabolized or further decomposed. The principal ions of cholesterol oxides monitored by LC/TS/MS were m/z 438 (cholestane triol) m/z 401 (cholesterol-7-hydroperoxide) m/z 401 (7-ketocholesterol) m/z 367 (7a-hydroxycholesterol) m/z 399 (cholesta-3,5-dien-7-one) and m/z 385 (choles-terol-5a,6a-epoxide). The major ions were supported by minor ions consistent with the steroid structure. Kamido et al. (1992a, b) synthesized the cholesteryl 5-oxovaleroyl and 9-oxononanoyl esters as stable secondary oxidation products of cholesteryl arachidonate and linoleate, respectively. These compounds were identified as the 3,5-dinitrophenylhydrazone (DNPH) derivatives by reversed-phase LC/NICI/MS. These standards were used to identify cholesteryl and 7-ketocholesteryl 5-oxovaleroyl and 9-oxononanoyl esters as major components of the cholesteryl ester core aldehydes generated by copper-catalysed peroxidation of low-density lipoprotein (LDL). In addition to 9-oxoalkanoate (major product), minor amounts of the 8, 9, 10, 11 and 12 oxo-alkanoates were also identified among the peroxidation products of cholesteryl linoleate. Peroxidation of cholesteryl arachidonate yielded the 4, 6, 7, 8, 9 and 10 oxo-alkanoates of cholesterol as minor products. The oxysterols resulting from the peroxidation of the steroid ring were mainly 7-keto, 7a-hydroxy and 7P-... 

In practice, eluents of widely varying polarity have been used. Very weakly polar steroids (oestrenols) can be quantitatively eluted with hydrophobic solvents like methylene dichloride (cf Table 14) [217], whereas difficulties are reported in the elution of the more polar progesterone under the same conditions [691]. Chloroform has been used to elute dinitrophenylhydrazones from silica gel and opium alkaloids from alumina [470, 535]. Methanol and ethanol are often used for elution of substances of all types of compound class from silica gel [213, 215, 259, 434] or alumina [122, 437]. Butyl acetate has been chosen as the most suitable eluent for penicillin V [486]. Acetone has proved suitable for recovering ubiquinones [733]. Polar neutral, acid and basic aqueous eluents have also been employed, e.g., water for mucic acid derivatives [451] (cf Table 14), 1% Tween 80 solution for cobalamin [128], 0.2 N sulphuric acid for Vitamin Bg factors [702], 34% ammonium persulphate solution for nicotinic acid [702] and ammonium hydroxide for azo dyes [638]. Nitro-4-acetaminophenetoles have been extracted from alumina with the highly polar dimethylformamide [489]. 

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